Mouse Interleukin-1 Beta

Introduction

Mouse IL-1ß (Interleukin-1 beta) is a cytokine that is produced in response to inflammatory signals, infections, and endotoxins. IL-1ß is initially produced as a precursor, which then undergoes intracellular cleavage by Caspase-1 to form the active cytokine. It is produced in many different cells such as monocytes, tissue macrophages, keratinocytes, and other epithelial cells. IL-1ß plays many roles as it involved in a variety of processes ranging from the immune response to carbohydrate metabolism. Dysfunction or dysregulation of IL-1ß is associated with conditions sepsis, rheumatoid arthritis, inflammatory bowel disease, neuronal injury, and many others.

Assay Overview

The workflow for the Pu•MA System cytokine immunoassay is shown in the figure below. All assay reagents are prepared in advance and then loaded into Pu•MA System flowchips using single or 8-channel pipettes. The wells are conveniently located on standard 384 multiwell plate spacings. The flowchips are now ready to be loaded into the Pu•MA System for “hands-off” processing. The ELISA steps are automatically performed by the system using pre-loaded protocols. Once the assay is complete, absorbance results are read on your Plate Reader.

  • Low-volume assays (10-20 μl)
  • Simple “hands-off” operation
  • Open platform – no expensive plates

Assay Procedure

The adaption of existing ELISA kits and/or antibody pairs is straightforward with the Pu•MA System. Assay buffers optimized for the Pu•MA System flowchips are provided for sample and antibody dilutions. Blocking solutions and wash buffers are also provided. The IL-1ß assays are adapted from BioLegend ELISA Max Kits (p/n 430901).

Assay reagents were prepared according to the dilutions shown in the Table using Pu•MA Assay Buffer (PAB). IL-1ß standards were reconstituted according to instruction provided by BioLegend. A 1:2 serial dilution series of Mouse IL-1ß Standard was prepared starting at 100 pg/well using PAB. 20 μl of each reagent was added to the appropriate wells (see Fig 3) except for the Stop Solution where 10 μl was dispensed. Four replicates were run per concentration. The flowchips were loaded into a Pu•MA System and processed using the IL-1ß Assay Protocol. Plates were read within 5 min of completion on an absorbance plate reader at 450nm (Tecan Spectrafluor Plus).

Pu•MA System Mouse IL-1β Standard Curve

Pu•MA System ELISA

PuMA System ELISA

How It Works

The PuMA Flowchip and System uses established antibody pairs to perform an automated ELISA. All assay reagents are loaded into reservoirs and then moved one at a time through the “Assay Channel” by the PuMA System. Preloaded protocols execute all fluid transfer and incubation steps The system incorporates patented valve-less fluidic switching (VLFS) to precisely control fluid movement in a flowchip. Use of microfluidics reduces both incubation times and reagent volumes.

Name Reagent Source
Capture Ab Mouse IL-1β Capture
Antibody (1:50)
BL
Block Pu•MA Blocking Buffer PFI
Sample Mouse IL-1β Standard BL
1°Ab Mouse IL-1β Primary
Antibody (1:50)
BL
2°Ab Avidin-HRP (1:5K) BL
Wash Pu•MA Wash Buffer PFI
Substrate FAST Substrate PFI
Stop Pu•MA Stop Solution PFI

Microfluidic Assay Workflow

Schematic of PuMA system ELISA

PuMA System

Pu•MA System

  • Compact benchtop system
  • Easy top-loading of flowchips
  • Precision pneumatic control

PuMA System Software

Software

  • Touchscreen-driven interface
  • Preloaded assay protocols
  • Simple Select and Run operation

Reagents and Flowchips

Reagents & Flowchips

  • Validated immunoassays
  • Open platform for existing Ab pairs
  • Works with standard pipettors & tips